Our goal is to develop novel fluorescence technology, reagents, and concepts, in order to discover new drugs for the treatment of heart failure (HF), the leading cause of morbidity and death worldwide. In HF, a key dysfunction is in Ca transport, which is needed to relax the muscle after each heart beat. A key source of this deficiency is declining activity of the sarcoplasmic reticulum Ca-ATPase (SERCA2a), which is regulated by phospholamban (PLB), a membrane protein that inhibits SERCA. For two decades, major drug companies have tried to develop small-molecule activators of SERCA or inactivators of PLB, but have failed. In this project, we introduce a series of technical and conceptual innovations to solve this problem. We seek to discover small-molecule compounds that disrupt the inhibitory SERCA-PLB interaction. Our approach is to conduct a high-throughput screen using fluorescence resonance energy transfer (FRET) to directly target the SERCA-PLB interaction, in an assay optimized to detect subtle allosteric structural changes required to disrupt inhibition. Based on a pilot screen, we conclude that conventional plate-readers, which measure fluorescence intensity, provide insufficient precision, resulting in high rates of false positives and negatives. Fluorescence lifetime detection could solve this problem, but commercially available lifetime plate readers have been too slow for high-throughput screening. Therefore, we have developed new technology for high- throughput fluorescence lifetime detection, 105 times faster than existing technology, and incorporated it into a novel plate reader. This instrument scans at the same rate as the best fluorescence intensity reader, but precision and resolution are increased by at least an order of magnitude. As a result, the useful information content of the initial screen is substantially improved, dramatically enhancing the speed and effectiveness of drug discovery. This revolutionary technology enables us to conduct a high-throughput fluorescence lifetime screen of the SERCA-PLB complex, to identify compounds that specifically activate Ca transport in the heart. Aims: (1) Optimize and validate the SERCA-PLB FRET assay for high throughput screening in a fluorescence lifetime plate reader. (2) Conduct the screen optimized in (1) on a 50K-compound library. (3) Perform secondary functional assays on hits selected in (2), in preparation for future efforts in medicinal chemistry and animal testing. This project is novel and exploratory, with high risk balancing against the potential for high impact, not only because of the new instrumentation, but also because we employ new classes of molecular constructs, involving integral membrane proteins in both reconstituted membranes and in live cells, never before used in a high-throughput screen. This project's high significance stems in part from the potential of compounds discovered here to revolutionize therapies for HF, as well as for other disorders linked to Ca homeostasis (e.g., cancer, diabetes, muscular dystrophy). Even greater impact will ensue if our approach establishes a new biophysical paradigm for fluorescence-based drug discovery.